Composition, preparation method thereof and application thereof in field of preservation of free DNA (deoxyribonucleic acid) in whole blood
Technical Field
The invention belongs to the technical field of nucleic acid preservation, and particularly relates to a composition, a preparation method thereof and application thereof in the field of preservation of free DNA in whole blood.
Background
Since the first discovery of free DNA in 1948, with the development of molecular detection technology, it was found that the level and composition of free DNA are closely related to various diseases. Free DNA is used as a biomarker, is gradually applied to various fields of early diagnosis, prognosis and monitoring of diseases, and is especially the most mature in the fields of tumor diagnosis and noninvasive prenatal screening. Free DNA in isolated blood is influenced by various factors such as macrophage elimination, enzyme degradation, release of nucleated cells and the like, and the quantity and the type of the free DNA change in the sample storage process, so that the accuracy of the detection result is influenced.
In order to reduce the isolated variation of free DNA to the maximum extent and reduce the storage and transportation cost, a special preservation solution is required in the application process, and the following three functions are performed: 1. inhibiting the activity of DNA enzyme and protecting free DNA from degradation; 2. inhibiting macrophage activity, and preventing macrophage from removing free DNA; 3. maintain the integrity of cells, reduce the apoptosis and necrosis of cells, and prevent the release of genome DNA, resulting in background pollution. In the prior art, EDTA anticoagulant plasma which is commonly adopted at present is used for extracting free DNA, and strict storage conditions are provided for temperature and storage time, so that the clinical use is greatly limited.
Therefore, the development of a composition, a preparation method thereof and an application thereof in the field of preservation of whole blood free DNA is used for solving the technical defects that in the prior art, the free DNA has strict storage conditions for temperature and storage time, which greatly limits clinical application, and thus the problem to be solved by the technical staff in the field is urgent.
Disclosure of Invention
In view of the above, the invention provides a composition, a preparation method thereof and an application thereof in the field of preservation of whole blood free DNA, and aims to solve the technical defect that in the prior art, free DNA has strict storage conditions for temperature and storage time, and clinical application is greatly limited.
The invention provides a composition, which comprises the following raw materials: permeability protectants, stabilizers, ion channel blockers, and anticoagulants.
Preferably, the raw materials of the composition comprise, by mass: 0.05-1 part of permeability protective agent, 5-50 parts of stabilizing agent, 1-10 parts of ion channel blocking agent and 8-16 parts of anticoagulant.
Preferably, the raw materials of the composition comprise, by mass: 0.1 part of permeability protective agent, 20 parts of stabilizing agent, 5 parts of ion channel blocking agent and 10 parts of anticoagulant
Preferably, the osmoprotectant is DMSO and/or glycerol.
Preferably, the stabilizing agent is inositol and/or trehalose.
Preferably, the ion channel blocker is selected from: one or more of tetraethyl amine, 4-aminopyridine, quinidine, quinine, barium chloride, DIDS, and NPPB.
Preferably, the anticoagulant is dipotassium ethylenediaminetetraacetate and/or tripotassium ethylenediaminetetraacetate.
The invention also provides a preparation method of the composition, which comprises the following steps:
mixing a stabilizer, an ion channel blocker and an anticoagulant to obtain a first mixture, adding water and stirring to obtain a first product;
and step two, mixing the first product with a permeability protective agent to obtain a second mixture, adding water, stirring, and filtering through a filter membrane to obtain a product.
Preferably, in the first step, the mass feed ratio of the first mixture to water is (17-64): (60-90);
in the second step, the volume feeding ratio of the second mixture to water is (4-12): (1-4).
The invention also provides an application of the composition or the product prepared by the preparation method in the field of preservation of free DNA in whole blood.
In summary, the present invention provides a composition, which comprises the following raw materials: permeability protectants, stabilizers, ion channel blockers, and anticoagulants. The invention also provides a preparation method of the composition and application of a product obtained by the preparation method in the field of preservation of free DNA in whole blood. The product prepared by the technical scheme provided by the invention can effectively stabilize blood cell membranes and reduce the rupture of red blood cells and white blood cells under the experimental condition of simulated transportation, and simultaneously has good protection effect on free DNA of whole blood, and further ensures the accuracy and reliability of detection results when the free DNA of whole blood is used for clinical detection. The composition, the preparation method thereof and the application in the field of preservation of whole blood free DNA provided by the invention solve the technical defects that in the prior art, the free DNA has strict storage conditions for temperature and storage time, and the clinical application is greatly limited.
Detailed Description
The embodiment of the invention provides a composition, a preparation method thereof and application in the field of preservation of whole blood free DNA, which are used for solving the technical defect that in the prior art, the free DNA has strict storage conditions for temperature and storage time, and the clinical application is greatly limited.
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In order to illustrate the present invention in more detail, the following examples are provided to describe a composition, a preparation method and applications thereof in the field of preservation of free DNA in whole blood.
Example 1
This example is a specific example of the preparation of a preservative product.
Weighing 10g of stabilizing agent, 2g of ion channel blocking agent and 10g of anticoagulant, mixing, adding 80mL of water, and uniformly stirring to obtain a first product 1; wherein, the stabilizing agent is inositol, the ion channel blocker is tetraethyl amine, and the anticoagulant is ethylene diamine tetraacetic acid dipotassium. Mixing the first product 1 with 0.1g of permeability protective agent, adding water to a constant volume of 100mL, uniformly stirring, and filtering by using a filter membrane with the pore diameter of 0.22 mu m to obtain a product 1; wherein the osmotic protective agent is DMSO.
Weighing 5g of stabilizing agent, 2g of ion channel blocking agent and 10g of anticoagulant, mixing, adding 60mL of water, and uniformly stirring to obtain a first product 2; wherein the stabilizing agent is trehalose, the ion channel blocking agent is 4-aminopyridine, and the anticoagulant is ethylene diamine tetraacetic acid dipotassium. Mixing the first product 1 with 0.1g of permeability protective agent, adding water to a constant volume of 100mL, uniformly stirring, and filtering by using a filter membrane with the pore diameter of 0.22 mu m to obtain a product 2; wherein the osmotic protective agent is glycerol.
Weighing 20g of inositol serving as a stabilizer, 2g of trehalose, 1g of an ion channel blocker and 8g of an anticoagulant, mixing, adding 90mL of water, and uniformly stirring to obtain a first product 1; wherein the stabilizer is a mixture of inositol and trehalose, the ion channel blocker is quinidine, and the anticoagulant is dipotassium ethylenediamine tetraacetate. Mixing the first product 3 with 0.05g of permeability protective agent, adding water to a constant volume of 100mL, uniformly stirring, and filtering by using a filter membrane with the pore diameter of 0.22 mu m to obtain a product 3; wherein the osmotic protective agent is DMSO.
Weighing 20g of stabilizing agent, 5g of ion channel blocking agent and 10g of anticoagulant, mixing, adding 80mL of water, and uniformly stirring to obtain a first product 4; wherein the stabilizing agent is inositol, the ion channel blocker is quinine, and the anticoagulant is ethylene diamine tetraacetic acid dipotassium. Mixing the first product 1 with 0.1g of permeability protective agent, adding water to a constant volume of 100mL, uniformly stirring, and filtering by using a filter membrane with the pore diameter of 0.22 mu m to obtain a product 4; wherein the osmotic protective agent is glycerol.
Weighing 50g of stabilizing agent, 2g of ion channel blocking agent and 12g of anticoagulant, mixing, adding 80mL of water, and uniformly stirring to obtain a first product 5; wherein the stabilizing agent is inositol, the ion channel blocking agent is barium chloride, and the anticoagulant is ethylene diamine tetraacetic acid tripotassium salt. Mixing the first product 1 with 0.1g of permeability protective agent, adding water to a constant volume of 100mL, uniformly stirring, and filtering by using a filter membrane with the pore diameter of 0.22 mu m to obtain a product 5; wherein the osmotic protective agent is glycerol.
Weighing 30g of stabilizing agent, 3g of ion channel blocking agent and 16g of anticoagulant, mixing, adding 80mL of water, and uniformly stirring to obtain a first product 6; wherein the stabilizing agent is inositol, the ion channel blocker is DIDS, and the anticoagulant is ethylene diamine tetraacetic acid tripotassium. Mixing the first product 1 with 1g of permeability protective agent, adding water to a constant volume of 100mL, uniformly stirring, and filtering by using a filter membrane with the aperture of 0.22 mu m to obtain a product 6; wherein the osmotic protective agent is glycerol.
Weighing 5g of stabilizing agent, 10g of ion channel blocking agent and 12g of anticoagulant, mixing, adding 80mL of water, and uniformly stirring to obtain a first product 7; wherein the stabilizer is trehalose, the ion channel blocker is NPPB, and the anticoagulant is ethylene diamine tetraacetic acid tripotassium salt. Mixing the first product 1 with 1g of permeability protective agent, adding water to a constant volume of 100mL, uniformly stirring, and filtering by using a filter membrane with the aperture of 0.22 mu m to obtain a product 7; wherein the osmotic protective agent is DMSO.
Example 2
This example is a specific example of the preservation effect of the product on free DNA in whole blood when the products 1 to 7 prepared in example 1 are used for simulated transportation.
Add product 1 ~ 7 respectively into 7 vacuum test tubes as the additive, gather same blood sample, wherein, the blood sample of group 1 adopts product 1 to preserve, the blood sample of group 2 adopts product 2 to preserve, the blood sample of group 3 adopts product 3 to preserve, the blood sample of group 4 adopts product 4 to preserve, the blood sample of group 5 adopts product 5 to preserve, the blood sample of group 6 adopts product 6 to preserve, the blood sample of group 7 adopts product 7 to preserve, the blood sample of group 8 sells conventional EDTA. K2 preservative solution and preserves. In each group, the volume ratio of the blood sample to the preservation solution was 50: 1.
Placing eight groups of blood samples stored in a storage solution on a shaking table horizontally, shaking at the temperature of 25 ℃ at the frequency of 60r/min, taking 1ml of blood samples respectively on the 0 th day (the day of blood collection), the 3 rd day after blood collection and the 7 th day after blood collection, separating plasma, observing the color of the plasma, and judging the hemolysis grade, wherein the hemolysis grade result of each sample is shown in table 1.
TABLE 1 statistical Table of hemolysis results
|
Group 1
|
Group 2
|
Group 3
|
Group 4
|
Group 5
|
Group 6
|
Group 7
|
Group 8
|
Day 0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
Day 3
|
1
|
0
|
0
|
0
|
1
|
1
|
1
|
3
|
Day 7
|
2
|
1
|
1
|
1
|
1
|
2
|
2
|
4 |
In table 1, 0 represents no significant hemolysis, 1 represents mild hemolysis, 2 represents moderate hemolysis, and 3 represents severe hemolysis; in clinical trials, if the grade of hemolysis is less than or equal to 2, the clinical trial is considered acceptable. As can be seen from table 1, in the examples, groups 1 to 7, the samples were protected from hemolysis both on the day of blood collection and on days 3 and 7 after blood collection, and the protection effect on the samples was significantly better than that of group 8 on days 3 and 7 after blood collection. The blood samples stored in group 8 preservatives were already hemolyzed more severely at day 3 after blood collection and could not be used for clinical examination.
Meanwhile, 1ml of blood samples are taken at the 0 th day after blood collection (namely the day of blood collection), the 3 rd day after blood collection and the 7 th day after blood collection respectively, free DNA is extracted, and the copy number of LINE1 in blood plasma is detected by a real-time fluorescent quantitative PCR method, and specific experimental results can be referred to table 2.
TABLE 2 LINE1 copy number statistical Table (expressed in CT values)
|
Group 1
|
Group 2
|
Group 3
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Group 4
|
Group 5
|
Group 6
|
Group 7
|
Group 8
|
Day 0
|
19.001
|
18.931
|
19.045
|
19.189
|
19.154
|
19.039
|
18.974
|
19.034
|
Day 3
|
18.918
|
19.069
|
19.374
|
19.046
|
19.272
|
19.375
|
19.214
|
17.319
|
Day 7
|
18.736
|
19.186
|
19.174
|
19.180
|
19.377
|
18.855
|
18.992
|
16.425 |
As can be seen from table 2, in the examples, groups 1 to 7, the samples were protected from the rupture of nucleated cells both on the day of blood collection and on days 3 and 7 after blood collection, and the protection effect on the samples was significantly better than that of group 8 on days 3 and 7 after blood collection. The group 8 preservative stored blood samples had significantly increased LINE1 copy numbers (decreased CT values) at days 3 and 7 after collection, indicating that there was a significant amount of nucleated cell damage, genomic DNA was released into the plasma, raising the background, resulting in a decreased free DNA proportion, interfering with detection.
By combining the experimental results obtained in tables 1 and 2, under the condition of simulated transportation, the product prepared by the technical scheme provided by the invention can effectively stabilize blood cell membranes, reduce the rupture of red blood cells and white blood cells, and further ensure the accuracy and reliability of the detection result when the free DNA of whole blood is used for clinical detection.
Example 3
This example is a specific example of the DNA protective effect of the product on whole blood dissociation when the products 1 to 7 prepared in example 1 are used for simulated transportation.
Add product 1 ~ 7 respectively into 7 vacuum test tubes as the additive, gather same blood sample, wherein, the blood sample of group 1 adopts product 1 to preserve, the blood sample of group 2 adopts product 2 to preserve, the blood sample of group 3 adopts product 3 to preserve, the blood sample of group 4 adopts product 4 to preserve, the blood sample of group 5 adopts product 5 to preserve, the blood sample of group 6 adopts product 6 to preserve, the blood sample of group 7 adopts product 7 to preserve, the blood sample of group 8 sells conventional EDTA. K2 preservative solution and preserves. In each group, the volume ratio of the blood sample to the preservation solution was 50: 1.
Eight groups of blood samples preserved in the preservation solution are respectively mixed with the same and equivalent exogenous genes, then the mixture is horizontally placed on a shaking table, the shaking table is used for shaking at the frequency of 60r/min under the condition of 25 ℃, the plasma is separated respectively on the 0 th day (the day of blood collection) after blood collection, the 3 rd day after blood collection and the 7 th day after blood collection, the total amount of the plasma DNA is measured by utilizing the qubit3.0 after free DNA is extracted, in addition, the copy number of the exogenous genes in the plasma is detected by utilizing a real-time fluorescent quantitative PCR method, and the specific experimental results refer to the table 3 and the table 4.
TABLE 3 plasma DNA content (expressed in ng/200ul plasma)
|
Group 1
|
Group 2
|
Group 3
|
Group 4
|
Group 5
|
Group 6
|
Group 7
|
Group 8
|
Day 0
|
0.68
|
0.68
|
0.66
|
0.68
|
0.66
|
0.66
|
0.68
|
0.65
|
Day 3
|
0.66
|
0.64
|
0.64
|
0.67
|
0.67
|
0.63
|
0.67
|
1.66
|
Day 7
|
0.84
|
0.67
|
0.62
|
0.67
|
0.62
|
0.74
|
0.71
|
14.12 |
As can be seen from table 3, in the examples, groups 1 to 7, the total amount of sample plasma DNA was protected from significant changes on the day of blood collection, and on days 3 and 7 after blood collection, and the effect of sample preservation was far superior to that of group 8. In group 8, the total amount of plasma DNA of the stored blood samples was significantly increased on days 3 and 7 after the blood sampling, indicating that the influence of the genomic DNA released by the rupture of nucleated cells on the total amount of plasma DNA was dominant, and it could not be observed whether any dnase degraded the original free DNA in the samples.
TABLE 4 foreign Gene copy number statistics Table (expressed in CT values)
|
Group 1
|
Group 2
|
Group 3
|
Group 4
|
Group 5
|
Group 6
|
Group 7
|
Group 8
|
Day 0
|
17.514
|
17.652
|
17.723
|
17.351
|
17.535
|
17.285
|
17.031
|
17.434
|
Day 3
|
17.763
|
17.772
|
17.427
|
17.126
|
17.197
|
17.917
|
17.532
|
19.321
|
Day 7
|
18.214
|
17.893
|
17.901
|
17.34
|
18.325
|
18.023
|
17.328
|
20.893 |
As can be seen from Table 4, groups 1 to 7 in this example protected the sample plasma from the degradation of the foreign gene on the day of blood collection, and on days 3 and 7 after blood collection. However, the number of foreign genes in plasma was significantly reduced (CT value was increased) on days 3 and 7 after the blood sampling in the group 8 stored blood samples. The length of the exogenous gene is 132bp, the exogenous gene is a DNA sequence which is artificially synthesized, the exogenous gene can be used as a mimic of free DNA, and the same gene sequence cannot be released by the rupture of nucleated cells.
The results in Table 4 demonstrate that products 1-7 of the examples are capable of preserving free DNA in samples without degradation and perform better than conventional preservatives available on the market.
By combining the experimental results obtained in tables 3 and 4, under the condition of simulated transportation, the product prepared by the technical scheme provided by the invention can effectively ensure that the free DNA is not degraded, and further ensure the accuracy and reliability of the detection result when the free DNA of whole blood is used for clinical detection.
In summary, the present invention provides a composition, which comprises the following raw materials: permeability protectants, stabilizers, ion channel blockers, and anticoagulants. The invention also provides a preparation method of the composition and application of a product obtained by the preparation method in the field of preservation of free DNA in whole blood. The product prepared by the technical scheme provided by the invention can effectively stabilize blood cell membranes and reduce the rupture of red blood cells and white blood cells under the experimental condition of simulated transportation, and simultaneously has good protection effect on free DNA of whole blood, and further ensures the accuracy and reliability of detection results when the free DNA of whole blood is used for clinical detection. The composition, the preparation method thereof and the application in the field of preservation of whole blood free DNA provided by the invention solve the technical defects that in the prior art, the free DNA has strict storage conditions for temperature and storage time, and the clinical application is greatly limited.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.